Download Characterization of the unconventional myosin VIII in plant cells and

The Plant Journal (1999) 19(5), 555±567
Characterization of the unconventional myosin VIII in plant
cells and its localization at the post-cytokinetic cell wall
Stefanie Reichelt1,2,², Alex E. Knight1,³, Tony P. Hodge1,
Frantisek Baluska2,3, Jozef Samaj2,4, Dieter Volkmann2
and John Kendrick-Jones1,*
1
Structural Studies Division, MRC Laboratory of
Molecular Biology, Hills Road, Cambridge CB2 2QH, UK,
2
Botanisches Institut, UniversitaÈt Bonn, Venusbergweg
22, D-53115 Bonn, Germany,
3
Institute of Botany, Slovak Academy of Sciences,
DuÂbravska cesta 14, SK-84223 Bratislava, Slovakia, and
4
Institute of Plant Genetics, Slovak Academy of Sciences,
Akademicka 2, SK-95007, Slovakia
Summary
Myosins are a large superfamily of motor proteins
which, in association with actin, are involved in intracellular motile processes. In addition to the conventional
myosins involved in muscle contractility, there is, in
animal cells, a wide range of unconventional myosins
implicated in membrane-associated processes, such as
vesicle transport and membrane dynamics. In plant cells,
however, very little is known about myosins. We have
raised an antibody to the recombinant tail region of
Arabidopsis thaliana myosin 1 (a class VIII myosin) and
used it in immuno¯uorescence and EM studies on root
cells from cress and maize. The plant myosin VIII is
found to be concentrated at newly formed cross walls at
the stage in which the phragmoplast cytoskeleton has
depolymerized and the new cell plate is beginning to
mature. These walls are rich in plasmodesmata and we
show that they are the regions where the longitudinal
actin cables appear to attach. Myosin VIII appears to be
localized in these plasmodesmata and we suggest that
this protein is involved in maturation of the cell plate
and the re-establishment of cytoplasmic actin cables at
sites of intercellular communication.
Introduction
The myosins are a large superfamily of molecular
motors which generate movement and mechanical force
in ATP-dependent interactions with actin ®laments. On
Received 19 May 1999; accepted 7 July 1999.
*For correspondence (fax +1223 213 556; e-mail [email protected]).
²
Present address: Department of Biochemistry, Royal Holloway College,
University of London, London TW20 OBE, UK.
³
Present address: Department of Biology, University of York, York YO1 5DD,
UK.
ã 1999 Blackwell Science Ltd
the basis of their conserved head or motor domain
sequences, i.e. the highly conserved region containing
the ATPase and actin binding sites, the myosins can be
divided into at least 14 classes (designated I to XIV)
(Cope et al., 1996; Mermall et al., 1998). In addition, each
class of myosin contains tail domains which are
characteristic for each myosin and are believed to be
responsible for the speci®c subcellular localization and
function of these motors.
In unicellular systems and mammalian cells the
involvement of the conventional two-headed, ®lamentforming myosins (myosin IIs) is well established in
muscular contraction and cytoplasmic contractile events
such as cytokinesis. In addition, the so-called unconventional myosins of class I, V and VI are now relatively
well described; several of them have recently been
shown to be involved in various actin-based and
membrane-associated functions, which include vesicle
transport, cytokinesis and such specialized functions as
auditory perception (for recent reviews see BaÈhler, 1996;
Fath and Burgess, 1994; Hammer, 1994; Hasson and
Mooseker, 1995; Mermall et al., 1998; Mooseker and
Cheney, 1995; Titus, 1993).
In comparison to the mammalian and protozoan
myosins, little is known about these motors in higher
plants (for review see Asada and Collings, 1997;
Kendrick-Jones and Reichelt, 1999). The most prominent
intracellular motile event depending on the actomyosin
cytoskeleton in plant cells is cytoplasmic streaming, i.e.
the movement of vesicles and organelles in Chara
internodal cells and pollen tubes (for review see
Kuroda, 1990; McCurdy and Williamson, 1991;
Williamson, 1993). In Chara cells cytoplasmic streaming
occurs at rates of up to 60 mm sec±1 (about an order of
magnitude faster than the speed of the fastest muscle)
(Kachar and Reese, 1988). A recently isolated myosin
protein from Chara (Higashi-Fujime et al., 1995;
Yamamoto et al., 1995) moves muscle F-actin in vitro
motility assays at a similar velocity, strongly suggesting
that the isolated protein is the motor responsible for
cytoplasmic streaming. In Lily pollen tubes (Miller et al.,
1995; Tang et al., 1989), and in various tissues from
several plants (La Claire, 1991; Qiao et al., 1989; Turkina
et al., 1987; Vahey et al., 1982; Yokota et al., 1995),
biochemical and immuno¯uorescence studies using
heterologous myosin antibodies have detected myosinlike proteins. Most of these reports demonstrated spotlike anti-myosin labelling throughout the cytoplasm and
along actin ®laments, and this was interpreted as
555
556 Stefanie Reichelt et al.
showing the association of putative myosins with
various vesicles and organelles (Grolig et al., 1988;
Grolig et al., 1996; Heslop-Harrison and Heslop-Harrison,
1989; Miller et al., 1995). A more recent study has shown
heterologous anti-myosin staining associated with plasmodesmata in Chara cells (Radford and White, 1998).
However, none of these putative myosin proteins has so
far been cloned and sequenced to con®rm their myosin
identity.
The ®rst myosin gene to be cloned and sequenced
from a plant, Arabidopsis thaliana myosin 1 (ATM1)
(Knight and Kendrick-Jones, 1993), was placed by
phylogenetic analysis of its motor domain sequence
into a new class of myosins (class VIII). Since then a
further 10 plant myosin genes (from Arabidopsis,
Helianthus, Acetabularia and Chlamydomonas) have
been
cloned
and
sequenced
(Kinkema
and
Schiefelbein, 1994; Kinkema et al., 1994; La Claire et al.,
1995; Vugrek and Menzel, personal communication) and,
on the basis of their conserved motor domain sequences, have been placed into three new classes
(classes VIII, XI and XIII) (Cope et al., 1996) by phylogenetic analysis. Further analysis of their highly divergent
C-terminal tail sequences con®rms this classi®cation.
None of the plant myosins identi®ed thus far belong to
class I or II. The plant myosins in class XI are closely
related to the animal myosin Vs but are suf®ciently
different to be grouped into a separate class (Cope
et al., 1996). Furthermore, PCR screens and the
Arabidopsis genome sequencing project indicate the
existence of additional myosins in classes VIII, XI and
XIII (Kinkema et al., 1994; La Claire et al., 1995; Moepps
et al., 1993; Plazinski et al., 1997; Vugrek and Menzel,
personal communication).
The important point to emerge from this analysis is
that all the myosin genes identi®ed from plants belong
to unique classes which do not contain animal or
protozoan myosins. The motile processes in higher
plants and animals may be speci®cally different and,
in order to investigate the plant-speci®c localization and
function, we generated an antibody against a recombinant tail region of myosin ATM1. The immunolabelling
patterns in root cells from Lepidium sativum (cress) and
Zea mays (maize) indicate that this myosin VIII is
predominantly localized at the cell periphery where it
is preferentially associated with those plasma membrane regions involved in the assembly of new cell
walls. The immunogold and immuno¯uorescence
images reveal that this myosin is also associated with
cell-to-cell contact areas in root cells and appears to
accumulate speci®cally in plasmodesmata. These results
suggest the intriguing possibility that in plant cells
actomyosin-based forces are involved in the selective
`gating' of plasmodesmata.
Results
Immunological studies of myosin VIII in root tissue
preparations
To raise antibodies speci®cally against the Arabidopsis
thaliana myosin (ATM1), a class VIII plant myosin, we
expressed and puri®ed its tail region and used it as the
immunogen. This tail region appears to be unique and
consists of a short stretch of predicted a-helical coiled-coil
(amino acids 950±1019 containing about 10 heptad repeats) and a unique C-terminal region (residues 1020±
1166) (Knight and Kendrick-Jones, 1993) which has no
apparent homologies or recognisable motifs. The only
noticeable features are two clusters of serine residues and
a cluster of basic residues at its very C-terminus. For
expression, a vector of the pGEX series was modi®ed so
that fusion proteins with GST (Glutathione-S-transferase)
on their N-termini and a stretch of six histidine residues on
their C-termini could be expressed. This vector is especially useful when, as in this case, proteolytic degradation
of the expressed protein is a problem. Using the modi®ed
vector with two af®nity labels, intact fusion protein could
be prepared for antibody production.
For our immunological studies, we used root tissue from
Arabidopsis thaliana, cress (Lepidium sativum) and maize
(Zea mays) and coleoptile tissue from maize for Western
blots. Since cress and Arabidopsis belong to the same
dicot family (Brassicaceae), antibodies raised against the
Arabidopsis thaliana myosin 1 (ATM1) were expected to
cross-react with a myosin of the same class in cress and
this appears to be the case. We also found that we could
obtain the same immuno¯uorescence pattern in maize
roots.
To check the speci®city of the af®nity-puri®ed polyclonal
anti-ATM1 antibody, immmunoblot analyses were carried
out on subcellular fractions from cress root and maize
coleoptiles (Figure 1). In both cress roots (Figure 1A,a) and
maize coleoptiles (Figure 1B,b), the antibody cross-reacts
with a 130 kDa band in the microsomal membrane fraction
(Figure 1). This subcellular distribution indicates that the
intact 130 kDa myosin VIII is associated with the membrane
fraction, reminiscent of the membrane localization of the
myr 4 myosin in rat brain fractions (BaÈhler et al., 1994). In
our experience, many of the unconventional myosins in
animal tissues and tissue culture cells are preferentially
bound to cytoskeletal-membrane fractions. In cress roots
there is also a faint cross-reacting band at 170 kDa (Figure
1a) which may be the product of one of the class XI myosin
genes recently identi®ed by Kinkema and Schiefelbein
(1994). When protein extracts were rapidly prepared from
cress roots in the presence of a wide spectrum of protease
inhibitors and MgATP and subjected to immunoprecipitation with the anti-ATM1 antibody, a single band of
approximately 130 kDa was detected on Western blots
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
Unconventional myosin VIII
557
been comprehensively studied (e.g. Baluska et al., 1992;
Blanca¯or and Hasenstein, 1995).
The anti-ATM1 antibody strongly labelled the peripheral
region underlying the transverse cell walls in the roots of
all three plants (Figures 2b,d and 3b). In contrast and
acting as an internal control, the longitudinal walls in these
cells exhibited rather punctate myosin VIII labelling (Figure
2d,h). The labelling was most prominent in the region
containing meristematic and early post-mitotic cells which
were undergoing cell division. The strongest labelling was
found along newly formed cell walls which were in the
process of maturation.
Controls using either pre-immune serum or secondary
antibody alone or antibody to vertebrate non-muscle
myosin II (Drenckhahn et al., 1983) did not show any
staining in root cells (data not shown).
Myosin VIII localisation during mitosis
Figure 1. Immunoblot analysis of subcellular fractions of cress root and
maize coleoptiles using the anti-ATM1 antibody.
A, B and C are the Coomassie stained SDS-PAGE gels and a, b and c are
the corresponding immunoblots. A,a, cress root membrane protein
pellet; B,b, maize coleoptile membrane protein pellet; C,c, cress root
extract immunoprecipitated with anti-ATM1 antibody (hc = antibody
heavy chains (55 kDa) and arrow at 130 kDa = myosin VIII. aA and bB are
5±20% acrylamide gradient gels and cC is a 15% acrylamide gel).
(Figure 1c). The size of the immunoprecipitated protein is
in good agreement with the predicted molecular weight of
the ATM1 protein (131.2 kDa) (Knight and Kendrick-Jones,
1993). There was no corresponding Coomassie-stained
band on the SDS-PAGE gel (Figure 1C) indicating that the
amount of myosin VIII in these tissues is likely to be very
low. Puri®ed myosin VIII tail fusion protein when mixed
with anti-myosin antibody speci®cally blocked the Western
blot and immuno¯uorescent staining patterns.
Immunolocalization of myosin VIII along the transverse
cell walls in root tissue
For the immunolocalization studies, we used root tissue
from Arabidopsis, cress and maize seedlings. Root tissues
were preferred because they are more convenient to
handle and their simple architecture allows one to
consistently identify the various cell types undergoing
different developmental programmes. Virtually identical
antibody labelling patterns were obtained with root tissues
from all three plants (images from Arabidopsis are not
shown). Cress and maize roots were preferred: cress
because it is closely related to Arabidopsis and its roots
are more robust, and maize because its cytoskeleton has
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
The distribution of myosin VIII, actin and microtubules
during stages in the cell cycle in root meristems in higher
plants are shown in Figures 2 and 3 and are summarised in
the schematic drawings in Figure 6. At the onset of mitosis
(prophase), actin is localised as dense irregular networks
surrounding the enlarged, round nucleus (Figures 2a, 3a
and 6a). At this stage, myosin VIII labelling is concentrated
along the transverse cell walls and is sparsely distributed
along the longitudinal cell walls (Figure 2b). At the
beginning of metaphase, when the nuclear envelope
disintegrates, actin staining is concentrated along the
two opposing transverse cell walls with a few actin
®laments reaching towards the centre of the cell (Figures
2g and 6b). Myosin VIII labelling is more intense than in
prophase and is concentrated at the opposing transverse
cell walls, co-localizing with the actin ®laments (Figures 2h
and 6b).
The different stages in the cell cycle can be readily
assigned by following the distribution of microtubules and
mitotic chromosomes. At metaphase, when the chromosomes are aligned midway between the spindle poles,
dense bundles of kinetochore microtubules connect the
chromosomes to the poles (see SP in Figures 3g and 6d).
During telophase, in the early phragmoplast (P) region
where the new cell wall forms between the separating
daughter nuclei, intense anti-tubulin staining is seen as a
broad phragmoplast band in the centre of the newly
forming cell plate (Figures 3d and 6e). In late cytokinesis,
the phragmoplast P* forms a ring-like structure surrounding the newly developing cell plate (Figures 3d,g and 6f).
Myosin VIII staining does not occur in the callose-rich cell
plate region in the early (P) or in the developing late
phragmoplast (P*) (Figures 3c,f and 6f) but is con®ned
entirely to the opposing transverse cell walls which are
strongly stained (Figure 3c,f,h). The DAPI-stained chroma-
558 Stefanie Reichelt et al.
Figure 2. Immuno¯uorescence localization of myosin VIII and actin in maize root cells.
Cells double-labelled in (a) and (c) with a monoclonal anti-actin antibody and in (b) and (d) with anti-ATM1 antibody. In (e) and (g) root cells stained with
the anti-actin antibody are shown and (f) and (h) show cells predicted to be at the same stages stained with the anti-ATM1 antibody. Scale bars in all
cases are 10 microns. It was more dif®cult to obtain the same quality of myosin labelling in myosin/actin compared with tubulin/myosin double labelling
because the methanol treatment, which is required for the actin staining, badly affects the myosin staining. Therefore, to demonstrate actin and myosin
localization in all cell stages it was necessary to carry out single staining as well as myosin/actin double staining. In (a), smaller isodiametric cells
undergoing mitotic division show actin ®laments densely concentrated along the transverse cell walls, and in (g) the actin in interphase cells occurs as
®ne-®lamentous networks. In (b), the transverse walls of the root cells are prominently stained with the anti-ATM1 antibody, especially the newly formed
walls in G1 stage cells shown in (h) which co-localizes with the actin-staining. In (c) and (e), elongated cells with long actin ®lament cables traversing the
cells are shown; whereas in the same (d) or similar cells (f) only the transverse walls are prominently stained with the anti-ATM1 antibody where it colocalizes with the actin-staining.
tin in these cell stages is still slightly condensed (Figure
3e,i), and the shape and small size of the nuclei reveal that
these cells are in a late phase of cell division. Later, in G1phase (G in Figure 3), shortly after disintegration of the
phragmoplast microtubules (Figure 3d,g), extensive labelling of the myosin VIII was detected in this area (Figures
3c,f,h and 6g). In these cells the chromatin in the nuclei is
uncondensed (Figure 3e,i), and the phragmoplast microtubules have become integrated into the cortical microtubule network (Figures 3g and 6h). At this late G1 stage,
anti-ATM1 staining is thus very strongly concentrated
along or in the newly formed cell wall (Figure 3b,f,h) and
the actin ®laments are arranged in a regular network
reaching from the cell walls to the nucleus (Figures 2c,e, 3a
and 6c). It should be noted that during all these stages the
transverse cell walls are always highly labelled with antiATM1 antibody whereas the longitudinal walls show only
punctate labelling (Figures 2b,d,h and 3b).
In interphase cells, after completion of cell division, the
transverse cell walls are stained with anti-ATM1 antibody,
but less brightly than during mitosis (Figure 2d,f). Actin
staining of the same region shows a very prominent Factin array, with characteristically arranged actin ®lament
bundles (Figures 2c,e and 6i). The actin co-localizes with
the myosin VIII labelling along the transverse cell walls
and extends into the cell centre where the bundles appear
to separate into several ®ner ®laments surrounding the
nucleus. These ®ner central actin bundles are not labelled
with myosin VIII.
Association of myosin VIII with the plasma membrane
and plasmodesmata
1. Confocal microscopy. Optical sections through cress
root tips labelled with the anti-ATM1 antibody con®rmed
that the myosin VIII was localized along the whole
transverse cell wall, whereas the longitudinal walls show
only punctate labelling. An overlay of a phase-contrast
image and the ¯uorescent myosin-staining is shown in
pseudo-colour (Figure 4a). The myosin VIII labelling
pattern is densely punctate, with the highest density of
label in new cell walls in early G1, shortly after phragmoplast disintegration. An overlay of optical sections (Figure
4b) reveals that the punctate labelling pattern passed
through the whole cell wall area. Along the longitudinal
cell walls a less dense distribution of myosin VIII in distinct
patches can also be observed. In Figure 4(c), the myosin
VIII labelling in G1-phase cells is shown at a higher
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
Unconventional myosin VIII
559
Figure 3. Double-labelling immuno¯uorescence localization of myosin VIII and actin
or tubulin in maize root cells.
Double-labelling of roots cells with (a) antiactin and (b) anti-myosin. Double-labelling
of root cells with (c) anti-myosin, (d) antitubulin and (e) DAPI-staining. Cells are in
G1-phase (G), early phragmoplast (P) and
late phragmoplast (P*) stages. During
mitosis the opposing transverse cell walls in
early and late phragmoplast stages (P and
P*) are speci®cally labelled with anti-myosin
VIII (see c). Cells in early G1-stage (G),
recognizible by the smaller size of their
nuclei (see e, DAPI-staining), show antimyosin VIII staining along the newly formed
cell wall. Double-labeling of root cells with
(f) anti-myosin and (g) anti-tubulin. The
mitotic spindle (Sp) and early phragmoplast
(P) microtubules are stained with antitubulin antibody (see g), but no staining of
anti-myosin VIII occurs in the spindle or in
the phragmoplast (see g). In the G1-stage
(G) cells are strongly stained with antimyosin VIII along the newly formed cell
wall (see f). Double-labeling of root cells
with (h) anti-myosin and (i) DAPI-staining.
During mitosis in late metaphase (M), the
transverse cell walls are strongly stained
with anti-myosin VIII. The newly formed cell
wall (G) in G1 stages shows strong antimyosin VIII staining. Scale bar is 10 microns
in all micrographs.
magni®cation, revealing intense myosin VIII labelling of
the plasma membrane associated with the newly formed
cell wall.
2. Immunogold electron microscopy. To con®rm these
immuno¯uorescence images, we prepared ultra-thin frozen sections of cress roots and subjected them to indirect
immunogold labelling with our anti-ATM1 antibody. In the
electron microscope, labelling was detected along the
newly formed cell wall between two cells (arrows indicate
the 10 nm immunogold particles in Figure 5a,b). This cell
wall shows the features of a young cell wall after the
disintegration of the phragmoplast and before it loses its
`wavy' appearance, characteristic of the ®nal stages in the
maturation of the cell wall. The anti-ATM1/immunogold
labelling (10 nm particles) occurred in association with the
plasma membrane and at thin membranous structures
going through the cell wall which are thought to be
plasmodesmata. Other regions of the sections were
virtually free of any immunogold labelling.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
For a more detailed localisation of the myosin in the cell
walls, we used ultra-thin sections of maize roots embedded in LR White Resin which preserves the membrane
ultrastructure better than cryo-®xation. Transverse as well
as longitudinal cell walls possessing primary and secondary plasmodesmata showed characteristic anti-ATM1/immunogold labelling (Figure 5c,d,e). In young transverse
cell walls, plasmodesmata were seen decorated with
strings of gold particles (Figure 5c) and in longitudinal
cell walls, where secondary plasmodesmata were mostly
arranged in large pit ®elds, the immunogold particles were
especially enriched in this area (Figure 5d,e). The cytoplasm and other cell structures were very rarely labelled
when compared with the pit ®elds.
Discussion
We have performed the ®rst cytological characterization of
an unconventional myosin in higher plants using an
560 Stefanie Reichelt et al.
Figure 4. Confocal visualization of cress root
cells labeled with anti-myosin VIII antibody.
(a) An overlay of a phase-contrast image
and immuno¯uorescent anti-myosin VIII
image of cress root sections are shown in
false colours, where red is the phase
contrast image and green the ¯uorescence
image of myosin VIII staining. The bar is 6
microns. (b) Optical sections through an
anti-myosin VIII labelled cress root section.
The bar is 10 microns. The region framed in
(b) is shown in (c) at higher magni®cation.
The bar is 2.5 microns. An overlay of
several optical sections taken in 10 micron
steps shows that the transverse cell walls in
all growing cells are stained (see b) and
cells in G1-phase are especially strongly
labelled along the newly formed cell wall
(see c).
antibody speci®cally raised against a recombinant tail
region of the ®rst plant myosin gene to be cloned and
sequenced. This gene (ATM1) was isolated from a dicot
plant Arabidopsis thaliana (Knight and Kendrick-Jones,
1993). The predicted molecular weight of ATM1 is 131 kDa,
and it contains a motor domain (head), four IQ-motifs
predicted to bind calmodulin or light chains and a tail
domain with a predicted short coiled-coil region which
implies its dimerisation into a double-headed molecule.
The whole tail region of this myosin was expressed and
used as an immunogen to generate speci®c antibodies.
Immunoblot analysis using these anti-ATM1 antibodies
identi®ed an approximately 130 kDa band which is highly
enriched in the membrane fraction in cress and maize
tissues. These results suggest that the myosin VIII is
membrane-bound, possibly by its C-terminal globular
region. In this connection it is interesting that the animal
myosin Is and myosin Vs are known to be bound to
membranes (Mooseker and Cheney, 1995).
Immuno¯uorescence and immunoelectron microscopy
of cress and maize root tissues using the anti-ATM1
antibody showed that the myosin VIII is concentrated
along the plasma membranes at transverse cell walls both
in meristematic cells and in the post-mitotically growing
root cells. Cells in G1 phase are extensively stained along
their young transverse cell walls, whereas there is only
punctate staining along longitudinal cell walls. The
immunolocalization patterns show that myosin VIII is
concentrated along the transverse walls where F-actin is
attached perpendicularly. This suggests that myosin VIII
may have some role in the formation of new cell walls as it
has a less dense distribution along established side walls.
One key feature of the new end walls is that they are a
specialized zone of concentrated cell±cell contact where
plasmodesmata are formed at cell division. It is signi®cant,
therefore, that immunogold studies show myosin to be
located at these intercellular plant cell junctions.
In previous studies, myosin-like proteins from plants
have been characterized either with cross-reacting antibodies or by stimulation of their ATPase activity by F-actin
(for review see Asada and Collings, 1997; Kendrick-Jones
and Reichelt, 1999). Such studies have revealed a variety of
proteins, perhaps re¯ecting the diverse classes of myosins. One 240 kDa protein has been isolated from Chara
that moves muscle F-actin at 60 mm sec±1 in in vitro motility
assays which strongly suggests that this protein is the
motor responsible for cytoplasmic streaming. This myosin
when viewed in the electron microscope after rotary
shadowing (Yamamoto et al., 1995) has similar characteristics to a myosin V, i.e. it is a dimer of two 210±220 kDa
heavy chains with pear-shaped heads (motor domains)
and a slightly curled globular tail about 80 nm long. Thus
far it has not been possible to clone and sequence the gene
for this Chara myosin. The predicted molecular weights of
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
Unconventional myosin VIII
561
Figure 5. Immunoelectronmicroscopic localization of myosin VIII in cress roots.
(a) The cell wall in the centre of this
micrograph shows the features of a
`fenestrated sheet' structure indicating a
young cell wall after the disintegration of
the phragmoplast and before the cell wall
loses its `wavy' structure, indicating the
increasing deposit of cellulose and cell
matrix components. The 10 nm immunogold/anti-ATM1
antibody
particles
(arrowheads) occur associated with the
plasma membrane of the new cell wall and
at thin membranous structures and
plasmodesmata going through the wall.
Other regions of the sections were virtually
free of any gold particles. Scale bar is 1
micron. (b) The new cell wall is shown at a
higher magni®cation, scale bar is 1.5
microns. (c) A recently formed transverse
cross-wall in a cortical cell from the basal
part of the meristem. The immuno-gold
particles decorate the primary plasmodesmata formed during cytokinesis. (D) A
longitudinal section through a cell wall in a
cortical cell from the transition region. Note
the massive accumulation of immuno-gold
particles in secondary plasmodesmata
organized into pit-®elds. (e) A longitudinal
section through a cell wall in a cortical cell
from the apical part of the elongation
region.
Immuno-gold
particles
are
prominently associated with pit-®elds. Scale
bars for Figure 5(c,d,e) are 0.5 microns.
class VIII myosins is about 130 kDa and for class XI
myosins about 170 kDa. In higher plants, a 170 kDa myosin
has been biochemically isolated from lily pollen tubes
(Yokota and Shimmen, 1994) and an antibody to this
protein recognizes 170 kDa polypeptides in tobacco,
Tradescantia and Arabidopsis tissue (Yokota et al., 1995).
When heterologous anti-myosin antibodies have been
used to stain plant cells, they have mostly produced
punctate staining patterns throughout the cell which is
indicative of the labelling of organelles and vesicles
involved in membrane transport (Grolig et al., 1988;
Miller et al., 1995; Qiao et al., 1989). It can be concluded
that homologues of the myosin superfamily exist in higher
plants and are involved in a variety of membrane
associated processes. However, it is clear that the antimyosin VIII localization reported here has distinct differences from these previous studies. One of the striking
features of myosin VIII localization is that it becomes
concentrated in newly deposited cross-walls in contrast to
its sparse occurrence along side walls. This suggests that it
may be involved, not so much in the process of vesicle
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
transport and fusion which bring cell plate precursors to
the mid-line of the phragmoplast, but in those processes of
cell plate maturation. Indeed, myosin VIII labelling is found
most strongly when the actin and microtubules of the
phragmoplast have depolymerized, and it is important to
examine the stage-speci®c processes in which myosin VIII
may be implicated.
Several studies show that cell wall formation occurs in
two separate stages. For instance, caffeine is known not to
effect the formation of the phragmoplast cytoskeleton nor
the actual deposition of the central cell plate, but the later
stage of fusion with the mother wall and the conversion of
the callose-rich plate into a less ¯exible wall (Hepler and
Bonsignore, 1990; Valster and Hepler, 1997). Mineyuki and
Gunning (1990) have reviewed these later stages of cell
division and concluded that insertion and maturation
factors interact with the cell plate or pass into it from the
cortical division site. Callose (a major component of the
cell plate) is removed from cell plates soon after they
attach (Northcote, 1989) and pectic polysaccharides are
inserted (Moore and Staehelin, 1988; Samuels et al., 1995).
562 Stefanie Reichelt et al.
Figure 6. Schematic drawings of the distribution of myosin VIII, actin ®laments and microtubules in cells of the meristem and transition growth zone in the
root apex.
Cell walls and the underlying plasma membrane are represented as one grey line. The drawings represent the distribution of the cytoskeletal elements in
the median level of the cells, only in late G1/S-phase are cortical microtubules shown (as indicated). (a) In prophase, actin ®laments are distributed as
dense irregular networks surrounding the enlarged round nucleus (Figure 2a). Myosin VIII labelling is concentrated along the transverse cell walls and
sparsely distributed along the longitudinal cell walls (Figure 2b,d). (b) In metaphase, the nuclear envelope disintegrates and the chromosomes are
arranged along the equatorial plane. The F-actin at this stage forms a dense network of short ®laments on the two opposing transverse cell walls with a
few actin ®laments reaching towards the cell centre, but not into the equatorial plane (Figure 2g). Myosin VIII labelling is more dense than in prophase,
concentrated at the opposing cell walls, co-localizing with actin (Figure 2h). (c) In G1-phase, the newly formed plasma membrane and cell wall separates
the daughter cells. Actin ®laments are arranged in a regular network reaching from the cell walls to the nucleus (Figure 2e). The highest density of myosin
VIII labelling is located along the newly assembled cell wall (Figure 2d). The transverse cell walls are also strongly labelled with ATM1 antibody whereas
the longitudinal walls show only weak punctate labelling. The different cell cycle stages can be identi®ed by the distribution of microtubules and the
chromosomes. (d) At metaphase, when the chromosomes are aligned midway between the spindle poles, dense bundles of microtubules connect the
chromosomes to the pole (Figure 3g). (e) After separation of the chromosomes into daughter nuclei in late telophase, microtubules of the early
phragmoplast (P-MTs) are concentrated between the separating nuclei (Figure 3d).At this stage, numerous Golgi-derived vesicles accumulate at the
equatorial plate to initiate cell plate formation. (f) During late telophase/G1-phase, the late phragmoplast microtubules (P*-MTs) are concentrated as a
ringlike-structure surrounding the leading edges of the assembling cell plate (Figure 3d,g). Additional vesicles fuse with the phragmoplast, extending it
outwards and eventually the phragmoplast fuses with the plasma membrane, and the two cells separate. In (d±f) no myosin VIII staining was observed in
the mitotic spindle during the separation of the chromosomes or in the early or late phragmoplast (Figure 3c,f). However, the opposing transverse cells
walls are strongly stained with myosin VIII during these stages in the cell cycle, co-localizing with actin ®laments during metaphase. (g) In early G1-cells,
after phragmoplast disintegration and cell plate assembly, the ATM1 labelling becomes concentrated along the newly formed cell wall which separates the
daughter cells (Figure 3b,c). (h) Later in G1-phase, when the nuclear membranes have completely reformed around each daughter nucleus and the nucleoli
are visible (Figure 3e,i), the microtubules and the actin are arranged as dense networks interconnecting the centrally located nucleus with the cell
periphery. At this stage, the highest density of ATM1 labelling is located along the newly assembled cell wall (Figure 3c). (i) During interphase, when the
root cells elongate, the actin ®laments are extending from perinuclear sites towards the opposite transverse walls of the cell where they co-localize with
the myosin VIII (only two actin bundles in the median level are shown) (Figure 2c,d). These actin ®laments may form a transport pathway for Golgi-derived
vesicles carrying new cell wall components.
The appearance of myosin VIII coincides therefore with the
onset of these wall-consolidating processes. The protein
could bring the islands of membrane plate material
together or it could trigger the exocytosis of new cell wall
material. High concentrations of calcium±calmodulin are
found near the phragmoplast/cell plate (Hepler, 1994) and
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
Unconventional myosin VIII
it is possible that changes in the calcium levels at the
transition between plate deposition/maturation affect the
localisation and activity of myosin VIII. In Chara, a
polyclonal antibody raised against smooth and skeletal
myosin has been shown to label plasmodesmata (Radford
and White, 1998) ± a localisation pattern which agrees with
our previous results (Reichelt et al., 1997) and this paper.
In the early post-mitotic root cells of cress and maize the
transverse walls are intensely labelled with the antimyosin VIII and actin antibodies. This myosin antibody
does not decorate F-actin cables involved in cytoplasmic
streaming but it is interesting that myosin VIII is concentrated along those walls to which the longitudinal actin
cables are directed and attached. The cytoplasmic actin
cables largely depolymerize in cell division but streaming
is restored along the interphase array during early
postcytokinesis. Another possible role for myosin VIII
(which is detected in the membrane fraction) is that it
may be part of the system for anchoring or directing the
cytoplasmic F-actin cables. Since F-actin goes to (or
through) plasmodesmata (White et al., 1994), it has been
suggested to be involved in the intercellular movement of
certain viruses (McLean et al., 1995), proteins and mRNA
(Lucas et al., 1996). Such particle movement is most likely
to be an exaggerated form of normal directed cell
transport. It is interesting therefore that the density of
plasmodesmata is far greater in the transverse end walls
than in the longitudinal side walls (Juniper and Barlow,
1969). The movement of virus particles (McLean et al.,
1995; Waigmann and Zambryski, 1995; Zambryski, 1995),
and the fact that cytochalasin D enlarges the plasmodesmatal neck (White et al., 1994), indicates that there is a
gating mechanism which controls the size of the pore and
that this is likely to be under contractile regulation (Ding
et al., 1996; Overall et al., 1982; White et al., 1994). The
present study has shown that myosin VIII is localized at
plasmodesmata and, particularly since it is an actininteracting protein, is a strong candidate for being part of
any intercellular gating complex.
Experimental procedures
Materials
Analytical grade reagents were obtained from BDH Chemicals Ltd
(Poole, UK), Bethesda Research Laboratories Inc. (Gaithersburg,
MD, USA) and Sigma Chemical Co. Ltd (Poole, UK). Enzymes
supplied by New England BioLabs (Hertfordshire, UK) and
Promega Corporation (Madison, WI, USA) were used according
to the manufacturer's standard assay conditions. Radiochemicals
were supplied by Amersham International (Amersham, UK).
Oligonucleotides were synthesized on an Applied Biosystems
390B automated synthesizer (Applied Biosystems Inc., Foster City,
CA, USA) by Terry Smith (Laboratory of Molecular Biology,
Cambridge, UK).
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
563
Expression of recombinant ATM1 and antibody
preparation
The vector pGEX-H6 used for the expression of the tail region of
Arabidopsis thaliana myosin VIII (ATM1) is based on pGEX4T-3
(Pharmacia) with the addition of a C-terminal string of six
histidine residues. This allows the intact expressed protein to be
puri®ed using two speci®c af®nity tags by the so-called `af®nitysandwich' protocol (Binder et al., 1994). The vector was constructed by inserting a double-stranded oligonucleotide consisting of: 5¢-GGC CGC CAC CAT CAC CAT CAC CAT ±3¢ and 5¢-GGC
CAT GGT GAT GGT GAT GGT GGC ±3¢ into the NotI site of
pGEX4T-3. This oligonucleotide introduces the 6 His (H6)
sequence C-terminal to a regenerated NotI site such that if the
insert is correctly engineered this His-tag should remain in frame
with both upstream sequences and the downstream stop codon.
The ATM1 insert was produced by PCR using the intact ATM1
cDNA clone (3.851Kb) (Knight and Kendrick-Jones, 1993) as
template and the following oligonucleotides: 5¢-CGA ATT CCC
GGG TCG GAG GTG CCA AGA CAA ATG AGT TAG GTG AG ±3¢
and 5¢-GCC ACG ATG CGG CCG CTA TAC CTG GTG CTA TTT CTC
CTT CCC CAC CA ±3¢ as primers which also introduce NotI and
XmaI restriction sites (underlined). The PCR product (2952±3632 in
the ATM1 cDNA) containing the whole tail-region of ATM1
including the coiled-coil and the C-terminal globular regions
(amino acid residues 938±1166) was ®rst blunt-end cloned into
puc18, digested with HincII and transformed into E. coli JM101
cells. Positive clones were recognized by blue/white selection and
PCR screening and were sequenced. Clones which contained the
correct inserts were digested with NotI and XmaI and ligated into
the dephosphorylated expression vector pGEX-H6. The ATM1pGEX-H6 construct was expressed in E. coli BL21 (DE3) cells
(Studier and Moffat, 1986). Freshly transformed colonies were
inoculated into 2XTY medium containing 100 mg ml±1 ampicillin,
grown overnight at 37°C and then induced with 0.6 mM IPTG and
grown for a further 3 h at 37°C. The cells were harvested by
centrifugation at 5000 g and resuspended in PBS containing 1%
TritonX-100 and a battery of protease inhibitors (see section on
immunoprecipitation and Western blot analysis). The cells were
lysed in a French Press and the supernatant collected by
centrifugation at 12 000 g. The ATM1 was expressed as a soluble
fusion protein, GST-ATM1-H6, with a molecular weight of 54 kDa.
The supernatant was ®ltered through a 0.45 mm ®lter and applied
to a Glutathione(GT)-Sepharose 4B column and puri®ed
according to the manufacturer's instructions (Pharmacia P-L
Biochemicals Inc.). After application of the expressed protein,
the GT-Sepharose column was washed with at least four volumes
(volume of supernatant applied) of PBS containing 1% Triton X100 and the protease inhibitor cocktail and the bound fusion
protein eluted with 10 mM glutathione, 50 mM Tris±HCl, pH 8.0.
Further puri®cation of the fusion protein using the His-tag by Niaf®nity chromatography was performed under non-denaturing
conditions according to the manufacturer's instructions
(Novagen, Inc.).
The ATM1-GST fusion protein was further puri®ed by SDSPAGE on a 10% acrylamide maxi-gel and the major band cut out
and electroeluted. Three 100 mg aliquots of this fusion protein
were used for antibody production in rabbits, as described by
Harlow and Lane (1988). The resulting polyclonal serum was
af®nity-puri®ed for the localization and Western blotting studies
in two steps; ®rst using a glutathione-S-transferase (GST)-agarose
af®nity column to remove any anti-GST antibodies and then using
the recombinant ATM1 protein on immunoblots to purify the antiATM1 antibodies. For these immunoblots the recombinant
564 Stefanie Reichelt et al.
protein puri®ed on a GT-Sepharose column was separated on an
SDS-Page gel and transferred to nitrocellulose. The nitrocellulose
blot was stained with Ponceau red and the strips containing the
recombinant ATM1 were cut out, blocked with 5% non-fat dry milk
in TBS (Tris buffered saline) for 1 h and then incubated with the
undiluted polyclonal serum overnight at 4°C on a rotating wheel.
The nitrocellulose strips were washed repeatedly with TBS
containing 0.05% Tween 20 and the bound ATM1-antibodies
eluted by vortexing the strips for 30 sec in 0.2 M Glycine, pH 2.2,
and then quickly neutralizing with 1 M Tris±HCl, pH 7.6. The
speci®city of these puri®ed antibodies was checked on blots;
they strongly cross-reacted against GST-ATM1 fusion protein and
thrombin cleaved 28 kDa fragment (see below) but gave no
reactivity against GST even at low dilution. These antibodies
weakly cross-react with the tail regions of puri®ed chicken skeletal
and brush border cytoplasmic myosin IIs. To check the identity of
the expressed GST-ATM1 fusion protein it was cleaved with
thrombin to yield 26 kDa and 28 kDa fragments. N-terminal
sequencing of the 28 kDa fragment con®rmed that it was the tail
region of ATM1 and the commercially available anti-GST antibody (Pharmacia P-L Biochemicals Inc.) identi®ed the 26 kDa
fragment as GST.
For competitive blocking of the anti-ATM1 antibodies, soluble
myosin VIII tail fusion protein was expressed and puri®ed using
Ni2+-NTA-Agarose chromatography (Qiagen Ltd) in PBS.
Immunoprecipitation, subcellular fractionation and
Western blot analysis
To detect the ATM1 protein in cells by immunoprecipitation, fresh
2-day-old cress roots were homogenized in an ice-cold solution
containing 0.2 M KCl, 15 mM Tris±HCl, ph 7.2, 1 mM EDTA, 5 mM
DTT, 0.1% Triton X-100, and a broad range of protease inhibitors
(10 mg ml±1 aprotinin, 10 mg ml±1 chick ovalbumin trypsin inhibitor,
10 mg ml±1 N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE),
10 mg ml±1 tosyl-L-arginine methyl ester (TAME), 0.1 mM phenylmethylsulphonyl ¯uoride (PMSF), 10 mg ml±1 pepstatin, 10 mg ml±1
leupeptin (all obtained from Sigma) and 0.5 mM Pefabloc
SC
(AEBSF)
4-(2-Aminoethyl)-benzenesulphonyl
¯uoride
(Boehringer, Mannheim, Germany). The roots were ®rst homogenized in a Polytron Omnimixer (Kinematica, Luzern,
Switzerland), then in a Dounce homogenizer and the cell debris
removed by ®ltration through one layer of nylon mesh. ATP and
MgCl2 were added to the preparation to a ®nal concentration of
5 mM and 10 mM, respectively, and the cells lysed in a French
press under high pressure. A further aliquot of 5 mM MgATP was
added, and the preparation immediately centrifuged at 50 000 g
for 30 min. Two millilitres of the supernatant was incubated with
100 ml anti-ATM1 antibody for 1 h at 4°C and then 50 ml of ProteinA-Sepharose CL-4B (Sigma, P-3391) (swollen in PBS) was added
and incubated for a further 1 h at 4°C. The immunocomplexes
bound to the protein-A-beads were washed three times with 2 ml
of PBS and then ®nally drained with a syringe inserted directly
into the beads to completely remove the PBS. For SDS-PAGE
(Matsudeira and Burgess, 1978), 50 ml of sample buffer (2%SDS,
10% glycerol, 100mMDTT, 60mMTris±HCl, pH 6.8 and 0.001%
bromphenol blue) was added to the beads, heated to 85°C for
5 min, centrifuged and the supernatant loaded onto a gel.
The protein samples were run on SDS-PAGE 5±20% acrylamide
gradient gels using the method of Matsudaira and Burgess (1978)
and electrophoretically transferred to 0.1 mm nitrocellulose
(Schleicher & Schuell, Dassel, Germany) according to the method
of Burnette (1981). The nitrocellulose was blocked with 10% (w/v)
Marvel (non-fat dry milk) in PBS containing 0.3% Tween-20
(blocking buffer) before incubating with the anti-ATM1 antibody
diluted 1 : 500 in blocking buffer. The secondary antibody was
biotinylated goat anti-rabbit IgG(H + l) Vectastain, diluted 1 : 200
and the blot developed using the Vectastain ABC Peroxidase kit
(PK-4001, Vector Laboratories, Peterborough, UK). Developing
solution was 0.9 mg ml±1 DAB, 0.4 mg ml±1 NiCl2, 0.01%H2O2 in
100 mM Tris, pH 7.5.
To prepare fractions containing the membrane bound proteins,
plant material (cell cultures from Arabidopsis, cress roots and
maize coleoptiles) was homogenized (10 min in a mortar) in an
ice-cold solution of 250 mM Tris±HCl (pH 8.0), 25 mM EDTA,
330 mM sucrose, 5 mM DTT, 5 mM ascorbic acid and 1 mM
phenyl-methylsulphonyl ¯uoride (PMSF) followed by ®ltration
through one layer of nylon mesh. All subsequent steps were
carried out at 4°C. The post-mitochondrial supernatant (15 min,
4000 g; Biofuge 22R-Heraeus, Offenbach, Germany) was centrifuged for 50 min at 13 800 g (Biofuge 22R-Heraeus). The resulting
pellet was used as the membrane bound protein fraction. For
Bradford analysis the latter sample was resuspended in 200 ml of a
solution containing 10 mM MES/bis-tris-phosphate (BTP) pH 7.5,
5 mM EDTA and 20% glycerol.
SDS-PAGE was performed on these samples diluted in sample
buffer containing 3% SDS, 6% sucrose, 100 mM DTT, 60mMTris±
HCl, pH 6.8, and 0.001% bromphenol blue. The samples were
heated to 80°C for 10 min, centrifuged and the supernatant loaded
onto 8% gels. After protein separation, the gels were stained with
Coomassie blue or electrophoretically transferred to nitrocellulose using a transblot cell (Bio-Rad, MuÈnchen, Germany). For
immunostaining (at room temperature) the nitrocellulose membrane was pre-incubated with 4% BSA in TBS for 30 min, washed
for 30 min (three times for 10 min) in TTBS (TBS buffer containing
0.05% Tween-20) and incubated with anti-ATM1 antibody (diluted
1: 1000 in TTBS; control in TTBS). After washing in TTBS the
membrane was incubated for 1 h in TTBS containing the
secondary antibody (goat anti-rabbit IgG, whole molecule)
conjugated to alkaline phosphatase (Sigma A-9919; dilution 1:
100 000). Washing in TTBS was followed by alkaline phosphatase
detection using the fast-red staining protocol described by
Druguet and Pepys (1977).
Immuno¯uorescence and immunogold localization of
ATM1
The specimens for immuno¯uorescence microscopy were prepared by the method of Baluska et al. (1992). Brie¯y, 2-day-old
cress and maize roots were cut and ®xed in 4% formaldehyde in
MTSB (Microtuble Stabilizing Buffer) containing 50 mM PIPES,
5 mM MgSO4 and 5 mM EGTA, pH 6.9 for 1 h at room temperature.
The roots were dehydrated in a graded ethanol series (30% to 97%
ethanol in PBS) (PBS contained 0.14 M NaCl, 2.7 mM KCl, 6.5 mM
Na2HPO4, 1.5mMK2HPO4, 3 mM NaN3, pH 7.3) then incubated in
wax:ethanol (1 : 2, 1 : 1 and 2 : 1 steps) and ®nally in®ltrated in
100% Steedman's wax (PEG-400 distearate and 1-hexadecanol,
9 : 1(w/w)) at 37°C. The in®ltrated roots were then embedded by
allowing the wax to polymerize at room temperature.
Longitudinal sections 4 mm thick were cut with a microtome
(Jung, Heidelberg, Germany) and mounted on slides coated with
glycerol-albumen (Serva). The sections were dewaxed in 100%
ethanol, rehydrated in an ethanol series (97% to 30% ethanol in
PBS), washed with PBS and then ®nally with MTSB. If sections
were subjected to immunostaining with anti-actin antibody,
incubation in methanol at ±20°C for 10 min was required after
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
Unconventional myosin VIII
the ethanol series (Vitha et al., 1997). After methanol, the sections
were washed with PBS and then MTBS. For immunostaining the
sections were incubated with the af®nity-puri®ed anti-ATM1
antibody (diluted 1 : 200 in PBS with 0.1% BSA) or monoclonal
anti-actin (ICN, anti-actin, clone C4, cat. no. 69±100) or anti atubulin antibody (Amersham, N356) (both diluted 1 : 200 in PBSBSA) for 60 min at room temperature. After washing with MTSB,
the sections were incubated with ¯uorescein isothiocyanate
(FITC) conjugated anti-rabbit IgG raised in goat (Sigma
Chemical Co, St. Louis, MO, USA, F-6005) or rhodamineconjugated anti-mouse IgG raised in goat (Sigma, T-5393), both
diluted 1 : 200 in PBS-BSA, for 1 h at room temperature. Controls
included incubating with secondary antibody only, mouse preimmune serum and monoclonal anti-thymus-myosin II antibody
which cross-reacts against a wide spectrum of cytoplasmic
myosin IIs in animal cells (Drenckhahn et al., 1983). DNA was
labelled by incubating with DAPI (10 mg ml±1) for 5 min at room
temperature. Specimens were examined in a Zeiss Axiovert using
a Zeiss Planachromat or Neo¯uar 403 or 1003. Micrographs were
taken using Kodak Ektachrome 400 ASA ®lm. The labelled
sections were also viewed in a confocal laser scanning microscope (Bio-Rad MRC 600) equipped with argon/krypton lasers and
dual excitation/emission ®lter sets for both TRITC and FITC for the
double-labelled specimens.
For immunoelectron microscopy 2-day-old cress roots were cut
and ®xed in 8% formaldehyde and 0.1% glutaraldehyde in PBS for
1 h at room temperature, embedded in a drop of 3.2 M sucrose and
frozen on metal holders in liquid nitrogen. Ultra-thin sections
were cut on an cryo-ultramicrotome (Ultracut UCT, Leica/ReichertJung, Germany) ®tted with cryodevice FC-S and transferred onto
nickel grids. The sections were blocked against non-speci®c
labelling by incubating in 0.02 M glycine in PBS for 10 min, 2%
gelatin in PBS for 5 min and 0.1% BSA (typeV, Sigma) +5% FCS in
PBS for 10 min. For immunolabelling the sections were incubated
at room temperature for 30 min with the primary antibody,
af®nity-puri®ed anti-ATM1 (diluted 1 : 50 in PBS/BSA). They were
washed with PBS/BSA and then incubated with secondary
antibody goat antirabbit IgG-10 nm gold conjugate (BioCell,
Cardiff, U.K) diluted 1 : 50 in PBS/BSA. The sections were washed
with PBS and the reaction stabilized with 1% glutaraldehyde in
PBS. After washing in distilled water, sections were contrasted
with ice-cold 0.4% uranyl acetate in 1.8% methylcellulose.
Labelled sections were examined in a Philips EM301 electron
microscope at 80 kV.
For the immunolocalisation studies on plasmodesmata (Figure
5c,d,e) a similar protocol was used. Two-day-old maize roots were
cut and ®xed in 4% paraformaldehyde in MTSB buffer for 90 min
at room temperature. After washing in MTSB and PBS, and
dehydration in a graded ethanol series, the tissue was embedded
in LR White Resin (Hard grade, Biocell, Cardiff, UK) and left to
polymerise at 36°C. Ultra-thin sections were cut on an ultramicrotome and transferred onto formvar coated nickel grids. The
sections were blocked with 50 mM glycine, 5% BSA and 5%
normal goat serum in PBS for 30 min and washed with wash
buffer (WB) (PBS containing 1% BSA and 0.1% gelatin). They were
incubated ®rst with anti-ATM1 (diluted 1 : 50 with WB) at room
temperature for 90 min, washed with WB and incubated with the
second antibody, goat anti-rabbit IgG-10 nm gold conjugate
(diluted 1 : 50 in WB) for 90 min. The sections were washed with
WB and PBS, post-®xed with 3% glutaraldehyde for 15 min,
washed extensively with distilled water and contrasted with icecold 2% aqueous uranyl acetate and 1% osmium tetroxide (to
enhance plasmodesmata visualization). The labelled sections
were examined in a Zeiss EM 10 electron microscope at 60 kV.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 555±567
565
Acknowledgements
We wish to thank Drs Paul Luzio and Nick Bright (Department of
Clinical Biochemistry, University of Cambridge) for the use of
their facilities and for advice and assistance with the immunoelectron microscopy; Pat Edwards and Dr Brad Amos for their help
with the confocal microscopy; Dr Jamie Cope for his advice on the
molecular biological aspects of this work; and Monika Polsakiewicz for excellent technical assistance. This work was supported
by fellowships to S.R. from GraduiertenfoÈrderung des Landes
Nordrhein-Westfalen, Deutscher Akademische Austauschdienst
(DAAD) and Boehringer Ingelheim Fonds. Financial support to
AGRAVIS (Bonn) by the Deutsche Agentur fuÈr Raumfahrtangelegenheiten (DARA, Bonn) and the Ministerium fuÈr Wissenschaft
und Forschung (MWF, DuÈsseldorf) is gratefully acknowledged.
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